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Chromatography- Jafar Hasbullah

presentation 22. 5 . 2013

Bent Neck

on 8 September 2013

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Transcript of Chromatography- Jafar Hasbullah

Chromatography Thin Layer Chromatography Paper Chromatography Column Chromatography HPLC Gas chromatography Affinity Ion exchange It’s not used in industrial / large scale methods
Because it’s inefficient . Can be used to seperate Proteins
Drugs from blood/ urine

Used in forensics Paper Chromatography can separate minute amounts of substances Acending Decending The structure of silica gel Judge the purity of a synthesized compound
Indicate the extent of progress of a chemical reaction
seperation of molecules The Stationary phase Silica Gel
Alumina coated into a piece of glass The surface of the silica is very polar due to the OH groups the seperation occours in TLC according to the polarity The seperation More polar substances have less retention values . so they move less than less polar molecules Applications of TLC Visualizing the TLC Plates Florecence added stationary phase
dipped / sprayed in a solution which will react and give a colour change -- Amino acids can be visualized by spraying ninhydrin
UV active substances can be observed under UV lamp (Non coloured compounds) purification technique protein
A small spot of solution containing the sample is applied to a plate(about 1.5 centimeters from the bottom edge)

small amount of an appropriate solvent (elutant) is poured in to a glass beaker

A strip of filter paper is put into the chamber

The container is closed with a cover glass

The solvent moves up the plate by capillary action The Method TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. Stationary phase is very polar ! Lutein - have OH polar so its more attracted to the stationary phase Seperation is governed by the polarity SIZE—Gel filtration

CHARGE—Ion exchange


VOLATILITY - Gas Molecules can be separated according to its The Procces Applications The Method Protein mixture applied to column

Solvent (buffer) applied to top, flowed through column

Different proteins interact with matrix to different extents, flow at different rates

Proteins collected separately in different fractions Sample is vapourized Transfered to the column Introduction Used in the separation of mixtures .suited for separation of fairly volatile liquids Mobile phase - Gas (innert - He)

Stationary phase - can be a solid or
a liquid at high temperatures Carried with the mobile phase (He) Components are seperated according to Sample volatility - volatile analytes move faster
Analite polarity - polar analytes intract with the polar columns volatility ;Ability to Vaporize Partition Chromatography gas chromatography can quantitatively determine materials present at very low concentrations Applications GC is widely used by forensic scientists –

Pollution studies from analysis of body fluids for the presence of illegal substances, to testing of fiber and blood from a crime scene, and to detect residue from explosives It is often used to test hazardous waste sites for determining personal protective equipment (PPE) level and emergency response testing. seperated in the colomn accoring to their volatility/ polarity Detected in the data system

1. Very good separation
2. Time (analysis is short)
3. Small sample is needed - ml
4. Good detection system
5. Quantitatively analyzed
6. High sensitivity Gel filtration GC Flow Chart Disadvantages
1. It can be used as a preparative technique because we can't apply a large sample quantity;

2. It can't be used in quantitative analysis;

3. and doesn't allow the separation of complex mixtures.

4.this is one of oldest method Method The Stationary phase- the column The gel Reading - After Detection in which the various solutes of a solution are allowed to travel down an absorptive column, the individual components being absorbed by the stationary phase The most popular method for the purification of proteins and other charged molecules Introduction In cation exchange chromatography positively charged molecules are attracted to a negatively charged solid support Conversely, in anion exchange chromatography, negatively charged molecules are attracted to a positively charged solid support. Advantages of GC The seperation SIZE



CHARGE Chromatography is the physical separation of a mixture into its individual components. chromatography The chromatography data system can be calibrated according to the compounds retention times Constant ! Chromatography is used to separate compounds according to its difference in retention value (Rf) Principle of chromatography Analytical technique used to get qualitative and quantitative data

Used in – analysis of pharmaceuticals
food products
Industrial chemicals High performance liquid chromatography Heart of all HPLC instruments
Separation of components happens here
Interaction of Stationary phase –Mobile phase –Sample The Column Mobile phase Stationary Phase The Column (To withstand the high pressure ) Cont ... High pressure ! Compression fittings
(leak free connection ) Pores in micrometers (1.5-10um) HPLC instruments Stationary phase The mobile phase liquid is propelled through the column
(stationary phase ) to the waste Mobile Phase To keep the mobile phase at a steady flow rate under pressure we need a pump .  high pressures of up to 400 atmospheres Next we need a way to inject the sample into this for this we have the injector Small amount of samples (few micro liters ) We have to do this without altering or stopping the flow of mobile phase . Thus maintaining the system pressure And there are other types of detectors as well … Not all compounds absorb UV light 2 MS . Mass spectrometer Absorbance is proportional amount of the components 1. UV detectors Chromatography data system An extra mobile phase To maintain a constant Temperature An extra Mobile phase (gradient analysis) All air should be removed from the mobile phase for optimum performance Mikhail Tswett (1872-1919) He continued to work with chromatography in the first decade of the 20th century
primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls
New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes. Separation eluent eluate Chromatography Jafar hasbullah 1.Stationary phase
2.Mobile Phase
3.Sample Q . Highly volatile liquids ? Q. How do we know which gas represents which points ? Q. How the separation occurs ? Q. how will the separation be ? Summary ... Thank you Q. How does the process occur ? attraction bit.ly/119oELg
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